Lentiviruses or lentiviral vectors derived from them are popular as gene delivery vehicles.
The lentiviral genome includes three genes found in retroviruses, namely gag, pol and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes internal structural proteins such as matrix, capsid and nucleocapsid proteins. The pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase (RT)), a protease and an integrase, and the env gene encodes the viral envelope proteins. The 5′ and 3′ LTRs serve to promote transcription and polyadenylation of the viron RNAs. Adjacent to the 5′LTR are sequences necessary for reverse transcription of the genome such as the tRNA binding site and for efficient encapsidation of viral RNA into particles such as the Psi site.
Lentiviral vectors require an envelope protein in order to transduce a target cell. The envelope protein is expressed in the cell producing the vector and becomes incorporated into the vector particle. These particles comprise a protein core expressed by the gag gene which encases the viral RNA. This in turn is encased by a portion of the cell membrane that contains the envelope protein.
A lentiviral vector packaging cell line must contain a gag gene to express gag protein, a pol gene to express the pol protein, a env gene to express the envelope protein and a rev gene to express the rev protein to bind to the rev-response element (RRE) on transcribed vector genome RNA containing the transgene to facilitate nuclear export for packaging into vector particles at cell surface.
In addition, some lentiviral packaging cell lines contain the tat gene to express tat accessory protein to enhance transcription. However, as current state-of-the-art lentiviral vectors are tat-independent and pseudotyped with heterologous envelopes, the only HIV genes needed for production are gag-pol and rev. In most cases, production of lentiviral vectors has been by transient transfection of 293T cells. Typically these vectors are pseudotyped with the VSV-G envelope, which has a broad tropism and is relatively stable. Transient transfection enables the production of high titers of lentiviral vectors. However, there are a number of limitations. These included a short period of high titer virus production that only lasts a few days and variability between titers in different batches of lentiviral vector. Additionally, there is a possibility of recombination between co-transfected plasmids, compromising the safety of the batches of virus produced. These factors make the stringent safety characterisation and scale up required for good manufacturing practice (GMP) challenging. Aside from these limitations, producing lentiviral vectors by transient transfection is expensive, and unless a more effective means of production is developed, the application of lentiviral vectors to the clinic is likely to be confined to the treatment of diseases with small numbers of patients and therefore low amounts of vector. For applications involving larger numbers of patients, for example β thalassaemia or applications involving direct vector injection such as vaccination, packaging cell lines stably expressing lentiviral vectors would provide a more efficient means of production. This would enable reproducible safety and efficacy batch characteristics, as well as opening new therapeutic options, such as injection of producer cells into patients, allowing long-term in-vivo therapeutic vector production.
There have been a number of challenges in making lentiviral packaging cell lines. Briefly, it has not been possible to achieve sufficient HIV gag-pol expression by stable transfection, which is thought to be a result of toxicity. Furthermore, the envelope most used to pseudotype lentiviral vectors, VSV-G, is also too toxic for stable expression in lentiviral packaging cell lines. Therefore many attempts to make lentiviral packaging cell lines have been inducible, most commonly using tetracycline-regulated promoters to control expression of HIV gag-pol, rev and VSV-G (3, 5, 6, 9, 10, 14, 18). Although an inducible cell line has been developed for clinical use for EIAV vectors (15, 16), none of the published reports of tetracycline regulated inducible packaging cell lines for HIV-based lentiviral vectors have reached clinical trials. The reasons for this involve low titers of stably transfected self-inactivating (SIN) lentiviral vectors, a relatively short period of vector production after induction and difficulties in scale-up.
A stable lentiviral vector packaging cell line, STAR, was previously developed through expressing codon-optimised HIV gag-pol, rev and tat from gammaretroviral vectors, with internal CMV promoters driving expression of the packaging components. Retroviral envelopes (including amphotropic MLV or RD114) were then expressed by stable transfection. Full LTR or SIN lentiviral vectors were introduced by transduction or stable transfection respectively (7). Transduction of STAR cells with full LTR lentiviral vectors led to titers of over 107 infectious units per ml, that were sustained for over 140 days in culture. Stable transfection of a SIN lentiviral vector led to lower titers in producer clones, but most of these were still over 105 infectious units per ml.
Based on calculations on the amount of lentiviral vector required for clinical trials, it is assumed that a packaging cell line preferably requires a titer of at least 105 infectious units per ml to be useful for producing lentiviral vectors for clinical trials. Thus, it is clear that STAR cells make a sufficient titer (even after stable transfection of a SIN lentiviral vector).
The construction of these STAR cells did provide important proof of principle that a stable lentiviral vector packaging cell line was achievable. However, there were a number of issues to be addressed before this work could be translated into clinical application. The cell line was constructed using an MLV vector with wild-type LTRs. Therefore, cross packaging of this vector resulted in HIV gag-pol into virions, which was transferred to cells transduced with vector. Furthermore, the 293T cells used were untraceable and therefore did not meet good manufacturing practice (GMP) guidelines. See the world wide web at ec.europa.eu/health/documents/eudralex/vol-4/index en.htm.